The present invention relates to chromogenic compounds which are useful as optical indicator compounds in analytical test systems. In particular, the present invention relates to novel chromogenic enzyme substrate compounds and their use in analytical test systems for the detection of enzymes in a liquid test sample.
The determination of enzymes is important in a variety of fields such as biochemical research, environmental and industrial testing, and medical diagnostics. The quantitation of enzyme levels in body fluids such as serum and plasma provides very useful information to the physician in diagnosing diseased states and their treatment. In addition to being analytes of interest in biological fluids, enzymes can also serve as detection reagents in a variety of analytical systems such as immunoassays and nucleic acid hybridization techniques. In such systems, enzymes are useful directly or indirectly as labels to monitor the extent of antigen-antibody binding or nucleic acid hybridization that occurs.
Accordingly, the desire to detect enzyme analytes and to use enzyme labels as a diagnostic tool in various analytical test systems has given rise to the development of optical indicator compounds for use in the detection and measurement of the activity of such enzymes. Typically, such known optical indicator compounds comprise a detectable chemical group, such as a fluorogen or a chromogen, which has been derivatized with an enzyme cleavable substrate group specific for the enzyme of interest. Such optical indicator compounds exhibit an optical signal which is different from the optical signal which is provided by the cleaved native form of the fluorogen or chromogen. In principle, the enzyme cleaves the indicator compound to liberate the fluorogen or chromogen in the form of a distinctly fluorescent or colored product to provide a change in fluorescence or color which is proportional to the amount of enzyme present which, in turn, can be correlated to the amount of analyte present in a liquid test sample.
In particular, the detection and/or determination of hydrolases, i.e., enzymes which catalyse hydrolysis reactions of esters, glycosidic bonds, peptide bonds, other carbon-nitrogen bonds, and acid anhydrides [see Lehninger, Biochemistry (Worth Publishers, Inc, New York, N.Y., 1970) p. 148], is of interest in the diagnosis and monitoring of various diseases such as, for example, the determination of amylase and lipase in the diagnosis of pancreatic disfunction [see Kaplan and Pesce, Clinical Chemistry--Theory, Analysis and Correlation (C. V. Mosby Co., St. Louis, Mo., 1984) Chapter 56], determination of N-acetylglucosaminidase (NAG) as an indicator of renal disease [see Price, Curr. Probl. Clin. Biochem. 9, 150 (1979)] and detection of esterase as an indicator for leukocytes [see Skjold, Clin. Chem. 31, 993 (1985)].
Enzymes have also gained importance in the diagnostic as well as the biotechnology fields. For example, alkaline phosphatase and .beta.-D-galactosidase have found increasing use as indicator enzymes for enzyme immunoassays [see Annals of Clinical Biochemistry 16, 221-40 (1979)]. Accordingly, the use of enzymes such as glycosidases, particularly .beta.-D-galactosidase, as indicator enzyme labels in analytical test systems has given rise to the development of substrate glycosides such as phenyl-.beta.-D-galactoside, o-nitrophenyl-.beta.-D-galactoside and p-nitrophenyl-.beta.-D-galactoside [see Biochem. Z., Vol. 33, p. 209 (1960)] which are hydrolysed by .beta.-D-galactosidase to liberate the phenols which are determined photometrically in the ultraviolet range, or the nitrophenols which are determined in the shortwave visible range, respectively. A few other examples are the chromogenic resorufin derivatives of European Patent application No. 156,347, and the chromogenic acridinone derivatives of European Patent Application No. 270,946.
The use of .beta.-D-galactosides has also been described in conjunction with histochemical investigations, such as the naphthyl-.beta.-D-galactosides described in Histochemie, Vol. 35, p. 199 and Vol. 37, p. 89 (1973), and the 6-bromo-.alpha.-naphthyl derivatives thereof described in J. Biol. Chem., Vol. 195, p. 239 (1952). According to such test systems, the naphthols which are liberated upon the interaction of the galactoside with the enzyme are reacted with various diazonium salts to yield the respective azo-dyes which can then be visualized.
There continues to be a need for new compounds having desirable combinations of chromogenic substrate properties such as extinction coefficient, absorbance maxima, water solubility, color shift, and turnover rate.
Merocyanine dyes have previously been used as analytical reagents, although not as chromogenic enzyme substrates. European Patent Application No. 47470 describes the use of cyanine and merocyanine dyes as labels for antibodies or antigens in an immunochemical assay. The labeled antigen or antibody is subjected to an immune reaction and contacted with a silver halide which is then exposed to light and developed. The resulting optical density is measured. PCT Publication No. 86-06374 describes a conjugate of a highly fluorescent merocyanine dye with a biologically active moiety useful in diagnostic assays. The application uses dye-labeled antibodies to measure analytes in a test sample. Kiciak [Roczniki Chemii 37,225(1963)] describes the preparation of a merocyanine acetate ester but gives no suggestion that it might function as a chromogenic enzyme substrate.